Methods of Quantifying MicroRNAs for Hypoxia Research: Classic and Next Generation

Significance: Recent evidence is uncovering the ever-increasing importance of microribonucleic acids (miRNAs) in the hypoxia response. In order to investigate the important roles that these small RNAs play, methods of quantification whether using classic single-gene methods or genome-wide technologies are necessary to obtain a global picture of the differential expression of miRNAs in hypoxia and their interplay with protein coding genes. Recent Advances: Building on the groundwork of classic quantitative polymerase chain reaction (qPCR) and microarrays, the advent of next-generation sequencing technology has revolutionized how small RNAs can be detected and quantified on a genome-wide scale and without a priori knowledge of the small RNA sequence. This method delivers accurate and comprehensive data on the expression and sequence of all expressed small RNAs, and the data can be further combined with other sequencing modalities to better understand miRNAs via integrated genomic analyses. Critical Issues: Advancing technology has increased the need for better methods of sample and library preparation and for bioinformatics tools. Speed, cost, sample input, and analysis expertise remain the mainstay critical issues of small RNA sequencing. Future Directions: Future hypoxia research will benefit from the application of genome-wide sequencing technologies. Analyses that combine genomic, transcriptomic, chromosome conformation, DNA/RNA-protein binding, and proteomics will help greatly advance hypoxia miRNA research.